Download I87 Infection, Haemorrhage and Death of Chick Embryos after

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J. gen. ViroL 0973), 2x, I87-I9I
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Infection, Haemorrhage and Death
of Chick Embryos after inoculation of Herpes Simplex Virus
Type 2 on to the Chorioallantoic Membrane
(Accepted 2I June I973)
SUMMARY
When the chorioallantoic membrane of the embryonated hen's egg was inoculated with herpes simplex virus type 2 strains, an infection resulted which spread
rapidly throughout the egg. Haemorrhage and death occurred in the embryo, and
haemorrhage was also observed in the chorioallantoic membrane. Virus was
recovered from the infected chorioallantoic membrane, allantoic fluid, amniotic
fluid and selected organs of the embryo. In contrast, similar inoculation of herpes
simplex virus type r strains gave no haemorrhage in chorioallantoic membrane or
embryos, embryos did not die and virus was recovered only from the inoculated
chorioallantoic membrane. Inoculation of either type into the allantoic cavity did
not result in spread o f virus to the embryo. Prolonged adaptation of herpes simplex
viruses to growth in eggs eventually resulted in spread of type I virus from the
chorioallantoic membrane to other regions of the egg and there were also marginal
increases in virulence of type 2 virus.
It is generally recognized that there are two types of herpes simplex virus (HSV), which
can be differentiated by various tests, one of which is the appearance of the pocks they
produce on inoculation on to the chorioaUantoic membrane (CAM) of fertile hen's eggs
(Hutfield, 1967; Parker & Banatvala, 1967; Nahmias et al. 1968). Preliminary observations
in this laboratory indicated another difference between type I and type 2 strains in pathogenicity for the whole egg. This was explored in detail with four strains of each type, of
which three were established laboratory strains and one was a fresh isolate. Type I laboratory
strains were H F E M , HIL, WAL and the fresh isolate was 22Ioi ; type 2 laboratory strains
were LOV, PAR, ~7x52, and the fresh isolate was 9889. An outline study was also made
with a further 2I HSV strains; nine strains were type I and twelve were type 2. Of the nine
type ~ strains, five had had several passages in tissue culture and four were fresh isolates;
of the twelve type 2 strains five had had several passages in tissue culture and seven were
fresh isolates. All strains were typed by pock characteristics on the CAM and appearance
o f c.p.e, in tissue culture; laboratory strains were also studied by the temperature marker
test (Longson, x971 ; Ratcliffe, I97I ). Five of the eight strains used in the detailed study
were supplied by Professor P. Wildy (Birmingham University) and had been further typed
by the kinetic neutralization test (Rawls et al. J968). Infectivities of the eight strains studied
in detail were assayed in human embryo lung cell cultures using the 5o % end-point dilution
method (Reed & Muench, ~938), titres varied from 2.I × IO° to 4"7 x io ~ TCDs0 per ml for
type I strains and from 2.I × IO5 to 3-2 × IO7 TCDs0 per ml for type 2 strains.
Fertile Leghorn hens' eggs were incubated at 36 °C and after I I days o-t ml undiluted
stock virus pool was inoculated on to the CAM which was exposed using the false air sac
technique. Great care was taken to ensure that during the drilling of the eggs the membranes
remained intact and any eggs showing signs of bleeding were discarded. Harvests of allantoic
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Table I. Spread of types I and 2 herpes simplex viruses in the egg
HSV type t
strains
Hr~EM ~- - 4
HIL ~
22IOI
WAL
LOV
Days of
c
~
~ ~__,.__,
harvest... I 2 3 6 I
2 3
CAM
Fluids
Allantoic
Amniotic
Embryo
Heart
Gut
Brain
+
+
+
+
,
6
t
HSV type 2 strains
~PAR
I715 z
z__~
c__~
__~
,
2 3 6 I
2 3 6 I
9889
~
2 3
.
6
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
-
+
+
+
+
+
+
-
+
+
+
+
+
+
+
-
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
--
+
+
+
+
+
+
+
+
+
+ = herpes virus recovered from harvests as indicated by pocks formed on the CAMs of eggs inoculated
with harvests.
- = herpes virus not recovered from harvests, no pocks formed on CAMs of eggs inoculated with
harvests.
fluid, C A M , a m n i o t i c fluid, heart, gut a n d b r a i n were m a d e I, 2, 3 a n d 6 days p o s t - i n o c u lation, t a k i n g the following p r e c a u t i o n s to prevent c r o s s - c o n t a m i n a t i o n . A syringe with
a fine gauge needle ( 2 6 G ) was inserted 4 to 5 m m into t h e b l u n t end o f the egg, a n d allantoic
fluid was w i t h d r a w n slowly. A f t e r carefully cutting a w a y t h e d r o p p e d region o f the C A M
with the shell still attached, the a m n i o t i c sac was lifted with b l u n t forceps a n d the a m n i o t i c
fluid was r e m o v e d with a syringe. T h e e m b r y o was r e m o v e d f r o m the a m n i o t i c sac b y carefully cutting the a m n i o t i c m e m b r a n e s so as to prevent c o n t a c t between the e m b r y o a n d
a l l a n t o i c fluid. T h e e m b r y o was w a s h e d free o f a m n i o t i c fluid in three changes o f I5o ml
n o r m a l saline, a n d cut l o n g i t u d i n a l l y to expose the h e a r t which was r e m o v e d together with
the h e a r t b l o o d . Samples o f liver a n d intestine were r e m o v e d at this stage. T h e b r a i n
was a s p i r a t e d using a syringe with a coarse gauge needle ( I 8 G ) , which was inserted into
the b a c k o f the h e a d between the eyes. T h e shell a n d C A M r e m o v e d earlier were s e p a r a t e d
to harvest the C A M .
A l l a n t o i c cavity i n o c u l a t i o n was p e r f o r m e d b y inserting a fine gauge needle u n d e r the
shell a n d directly i n o c u l a t i n g the allantoic fluid with o q ml u n d i l u t e d stock virus p o o l .
A l l a n t o i c fluid a n d b r a i n were harvested as described above.
Harvests were stored at - 7 o °C. Virus in the harvests was d e m o n s t r a t e d b y i n o c u l a t i o n
o f harvests on to the C A M s o f further eggs, a n d o b s e r v a t i o n o f the C A M for d e v e l o p m e n t
o f typical herpes p o c k s w i t h i n 3 days. A l l a n t o i c a n d a m n i o t i c fluids were i n o c u l a t e d undiluted, h e a r t a n d gut m a t e r i a l were i n o c u l a t e d as 4o % (v/v) suspensions in penicillin/
s t r e p t o m y c i n b r o t h saline (PSBS), b r a i n was i n o c u l a t e d as a 20 % (v/v) suspension in PSBS
a n d C A M was h o m o g e n i z e d a n d i n o c u l a t e d at a IO -3 dilution in distilled water.
E a c h strain p r o d u c e d p o c k s on the C A M typical o f the t y p e to which the strain belonged,
i.e. t y p e I p o c k s were less t h a n o. 5 m m in diam. a n d type 2 p o c k s were greater t h a n I m m
after 3 days. T y p e 2 always caused a h a e m o r r h a g i c c o n d i t i o n o f the C A M . N o h a e m o r r h a g e
was seen after i n o c u l a t i o n o f the C A M with type I strains. Three days after C A M i n o c u l a t i o n
with t y p e 2 strains, the e m b r y o s were also h a e m o r r h a g i c a n d often dead. N e i t h e r d e a t h n o r
h a e m o r r h a g e o f e m b r y o s occurred w h e n C A M s were i n o c u l a t e d with type I strains even
after p r o l o n g e d i n c u b a t i o n .
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Type I viruses were recovered only from harvests of CAM. All other regions of the egg
harvested after inoculation of type I viruses on to the CAM failed to yield pocks on further
passage in eggs, indicating that HSV type I had not spread beyond the CAM. Results were
identical for all type I strains on all days tested. (Table I).
In contrast, all type 2 strains tested spread rapidly from the CAM to other regions of the
egg, appearing in the allantoic fluid I or 2 days after CAM inoculation, in the amniotic
fluid 2 days after inoculation and in the embryo harvests 2 or 3 days after inoculation. All
selected parts of the embryo yielded virus. The results were identical for all type 2 strains
tested (Table 0. Similar results were obtained when HSV type 2 virus strain 9889 (fresh
isolate) was diluted m -1 to io -4 although progressive dilution resulted in delayed spread to
the embryo. When even higher dilutions of the type 2 strain were inoculated on the CAM,
so that inocula contained only 5 to 2o pock forming units, no haemorrhage of the CAMs
was observed, and no virus was recovered from the embryo. Under these conditions, embryos
hatched normally. In contrast spread of type I virus beyond the CAM was never demonstrated even using the stock virus pools undiluted. The titres of type I virus in these pools
were comparable with the titres of the type 2 virus pools.
Herpes simplex virus type x strain 22io~ and HSV type 2 strain LOV were examined
after 12 and 24 serial egg passages. The nature and rate of spread were unaffected with either
strain after x2 egg passages, but after 24 egg passages the type 2 strain spread more rapidly
to the organs of the embryo, appearing in the allantoic fluid and brain only I day after CAM
inoculation. The type I strain spread into the allantoic fluid after 4 days but did not spread
to the brain of the embryo.
Following inoculation of either type I or type 2 virus into the allantoic fluid, no virus was
recovered from the embryo brain. The amount of virus recovered from the allantoic fluid
declined with time.
Further studies with 2I other strains showed that all 12 type 2 strains spread from the CAM
to the allantoic fluid within 3 days, whilst none of the 9 type I strains had spread beyond
the CAM 3 days after inoculation.
These results show that type 2 herpes simplex viruses spread rapidly from the inoculated
CAM to other regions of the egg including the embryo, whereas type I viruses were restricted
to the inoculation site. The rate of spread of type 2 virus was related to the concentration of
infectious virus in the inoculum. Following inoculation with small concentrations of virus
spread was delayed and with even smaller amounts the CAM was not haemorrhagic and no
virus was recovered from the embryo which, apparently unaffected, hatched normally.
Inoculation of HSV type I in concentrations equal to or exceeding those of type 2 did not
induce spread beyond the CAM, indicating that restriction of type ~ virus to the inoculation
site was truly type specific and not concentration dependent.
There was evidence that frequent serial passages in eggs somewhat increased the spreading
potential of the type I strain. After z4 egg passages the type I strain had spread into the
allantoic fluid 4 days after CAM inoculation, although embryos were not infected even
6 days post-inoculation. Type 2 virus at the 24th egg passage had become even more pathogenic for the CAM, causing greater haemorrhage and necrosis. It was also more rapidly
pathogenic for the embryo, and the rate of spread to the allantoic cavity was also increased.
As observed by Nahmias et aL 0968), HSV type 2 infection of the CAM produced lesions
involving all three layers of the membrane, that is ectoderm, mesoderm and endoderm,
whilst HSV type I lesions were restricted to the outer ectoderm layer. It was interesting that
in this study type 2 HSV apparently passed through the CAM, perhaps infecting all three
germ layers on route, to reach the allantoic fluid. It could be recovered from the fluid in
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large quantities for at least 6 days, but virus inoculated directly into the allantoic fluid declined in concentration. This suggests that the cells lining the allantoic cavity are either
insusceptible, or are infected only with difficulty from the allantoic fluid side of the membrane.
Since virus inoculated into the allantoic fluid did not spread to the embryo it seems likely
that after inoculation of HSV type 2 on to the CAM, virus infection of the embryo is not
via the allantoic fluid, but is due to virus penetration of the blood vessels lying in the mesoderm layer of the CAM. Spread of virus to the embryo was always associated with haemorrhage of the CAM. When a small dose of HSV type 2 was inoculated onto the CAM few
pocks were produced, the CAM was not haemorrhagic, and there was little if any spread of
virus to the embryo. It is possible that spread was prevented because the few pocks formed
were remote from blood vessels. Anderson (I94o) using the HF strain and Wildy & Holden
(1954) using the multiple egg passaged derivative strain HFE observed death of embryos.
These observations were made prior to dividing HSV into two types and a comparative
study of HF strains obtained from several sources revealed that some cultures were type I
while others were type 2 (Dowdle et al. I967).
Anderson (I94O) showed that vascular spread of herpes virus within the egg was possible
by demonstrating herpes virus intra-nuclear inclusions in the endodermal cells lining the
embryonic blood vessels and by isolating virus from the allantoic artery 2 days after inoculation of the CAM.
The reason for the restriction of type I strains to the inoculation site on the CAM compared with the rapid spread of type 2 strains to other regions of the egg can at present only
be assumed to be due to the relatively superficial nature of the primary CAM lesion. It
appears that this property of invasiveness is reproducible and type specific, although caution
is necessary in interpreting results observed with strains which have had multiple egg passages.
Recovery of infectious virus from the allantoic fluid three days after inoculation of a herpes
strain onto the carefully preserved intact CAM, taken together with the appearance and
size of the CAM lesions produced, could prove a useful additional test in the differentiation
of type I from type 2 herpes simplex viruses.
The author wishes to thank Dr Hazel Appleton (nee Ratcliffe) who performed the temperature marker tests and supplied some of the strains used, and also Drs M. Butler (University of Surrey) and Anne M. Field, and Mr J. R. McDonald for valuable advice and
discussion throughout this work.
Virus Reference Laboratory
Central Public Health Laboratory
Colindale Avenue
London NW9 5HT, U.K.
F . G . RODGERS
REFERENCES
ANDERSON, K. (1940). Pathogenesis o f herpes simplex virus infection in chick embryos. American Journal of
Pathology x6, 137-156.
DOWDLE, W. R., NAHMIAS, A. J., HARWELL, R. W. & PAULS, F. P. (I967). Association o f antigenic type o f herpesvirus h o m i n i s with site o f viral recovery. Journal of Immunology 99, 974-980.
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PARKER, J. D. J. & BANATVALA,J. E. (I967). Herpes genitalis: Clinical a n d virological studies. British Journal
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RATCLIFFE, H. (1971). The differentiation of herpes simplex virus type I and type 2 by temperature markers.
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RAWLS, W. E., LAUREL,D., MELNICK,J. L., GLICKSMAN,J. M. & KAUFMAN,a. H. 0968). A search for viruses in
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(Received I 2 April 1973)
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